Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids

Abstract

Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

Keywords: Azide Vibrational Reporters; Infrared Spectroscopy; Unnatural Amino Acids; superfolder Green Fluorescent Protein.

Figure 1

Structure of the series of the azido-modified unnatural amino acids.

Figure 2

Structure of wt-sfGFP (PDB ID 2B3P) with site 150 (magenta) highlighted.

Figure 3

FTIR absorbance spectra of 3 (Panel A) and 4 (Panel B) dissolved in either DMSO or an aqueous basic solution (200 mM NaOH) at a concentration of ~50 mM.

Figure 3

FTIR absorbance spectra of 3 (Panel A) and 4 (Panel B) dissolved in either DMSO or an aqueous basic solution (200 mM NaOH) at a concentration of ~50 mM.

Figure 4

Coomassie blue stained tris-glycine SDS-PAGE illustrating UAA incorporation into sfGFP at site 150 with high fidelity. The protein constructs were expressed from pBad-sfGFP-150TAG and pDule- pN₃Phe (lanes 2 – 6) in the presence (lanes 2, 3, 4, and 5) or the absence (lane 6) of 1, 2, 3, or 4, respectively.

Figure 5

FTIR absorbance spectra of sfGFP constructs containing 1, 2, or 4 incorporated at site 150 dissolved in a pH 7.3 aqueous buffer containing 50 mM sodium phosphate and 150 mM sodium chloride at a concentration of ~1 mM.

Scheme 1